Kinetic Characterization of Ribonuclease-Resistant 2′-Modified Hammerhead Ribozymes

Abstract

The incorporation of 2′-fluoro- and 2′-aminonucleotides into a hammerhead ribozyme was accomplished by automated chemical synthesis. The presence of 2′-fluorouridines, 2′-fluorocytidines, or 2′-aminouridines did not appreciably decrease catalytic efficiency. Incorporation of 2′-aminocytidines decreased ribozyme activity approximately by a factor of 20. The replacement of all adenosines with 2′-fluoroadenosines abolished catalysis in the presence of MgCl 2 within the limits of detection, but some activity was retained in the presence of MnCl 2 . This effect on catalysis was localized to a specific group of adenines within the conserved single-stranded region of the ribozyme. The decrease in catalytic efficiency was caused by a decrease in the rate constant; the Michaelis constant was unaltered. The 2′-fluoro and 2′-amino modifications conferred resistance toward ribonuclease degradation. Ribozymes containing 2′-fluoro- or 2′-aminonucleotides at all uridine and cytidine positions were stabilized against degradation in rabbit serum by a factor of at least 10 3 compared to unmodified ribozyme.